Regulated bacterial lysis for gene vaccine vector delivery and antigen release

Abstract

The present invention generally relates to regulated host-vector systems for delivery of genetic vaccine vector or desired gene products to a eukaryotic host, preparations of bacterial genetic vaccines, genetically engineered microorganisms that are useful for delivery of genetic vaccine vectors or desired gene products, and methods of use for the host-vector system.

1 . A host-vector system, which comprises 
 a. A host chromosome comprising 
 i. An activatible control sequence, wherein the activatible control sequence is activatible by an inducer;  
 ii. a sequence that encodes a repressor, wherein the sequence is operably-linked to the activatible control sequence; and  
 iii. at least one essential gene, wherein the essential gene encodes a polypeptide that is necessary for synthesis of a rigid layer of a cell wall of a prokaryote, and wherein the essential gene is inactivated;  
 AND  
   b. A vector comprising 
 i. a eukaryotic expression cassette comprising 
 1. A eukaryotic promoter sequence;  
 2. A site for insertion of a gene encoding a desired gene product; and  
 3. A polyadenylation sequence;  
 
 ii. a prokaryotic activator-promoter sequence;  
 iii. at least one origin of replication (ori);  
 iv. a regulatable prokaryotic promoter, which is repressible by the repressor;  
 v. at least one essential gene, wherein the essential gene is necessary for synthesis of a rigid layer of a cell wall of a prokaryote;  
 vi. at least one transcription terminator sequence; and  
 vii. at least one CpG sequence motif, wherein the CpG sequence motif enhances immunogenicity.  
   
   
   
       2 . A host-vector system, which comprises 
 a. A host chromosome comprising 
 i. An activatible control sequence, wherein the activatible control sequence is activatible by an inducer;  
 ii. at least one sequence that encodes a repressor, wherein the sequence is operably-linked to the activatible control sequence; and  
 iii. at least one essential gene, wherein the essential gene encodes a polypeptide that is necessary for synthesis of a rigid layer of a cell wall of a prokaryote, and wherein the essential gene is operably linked to the activatible control sequence;  
 AND  
   b. A vector comprising 
 i. a eukaryotic expression cassette comprising 
 1. a eukaryotic promoter sequence;  
 2. a site for insertion of a gene encoding a desired gene product; and  
 3. a polyadenylation sequence;  
 
 ii. a prokaryotic activator-promoter sequence;  
 iii. at least one origin of replication (ori);  
 iv. a regulatable prokaryotic promotor sequence, wherein the regulatable prokaryotic promotor sequence is repressible by the repressor;  
 v. at least one essential gene, wherein the essential gene is necessary for synthesis of a rigid layer of a cell wall of a prokaryote;  
 vi. at least one transcription terminator sequence; and  
 vii. at least one CpG sequence motif, wherein the CpG sequence motif enhances immunogenicity.  
   
   
   
       3 . (canceled)  
   
   
       4 . A host-vector system, which comprises 
 a. A host chromosome comprising 
 i. an activatible control sequence, wherein the activatible control sequence is activatible by an inducer;  
 ii. at least one sequence that encodes a repressor, wherein the sequence is operably-linked to the activatible control sequence; and  
 iii. at least one essential gene, wherein the essential gene encodes a polypeptide that is necessary for synthesis of a rigid layer of a cell wall of a prokaryote, and wherein the essential gene is inactivated;  
 AND  
   b. At least one vector comprising 
 i. a prokaryotic activator-promoter sequence;  
 ii. at least one origin of replication (ori);  
 iii. a first regulatable prokaryotic promotor sequence, wherein the first regulatable prokaryotic promotor sequence is repressible by a first repressor;  
 iv. a second regulatable prokaryotic promotor sequence, wherein the second regulatable prokaryotic promotor sequence is repressible by a second repressor;  
 v. at least one essential gene, wherein the essential gene is necessary for synthesis of a rigid layer of a cell wall of a prokaryote;  
 vi. at least one transcription terminator sequence; and  
 vii. a site for insertion of a gene encoding a desired gene product.  
   
   
   
       5 . (canceled)  
   
   
       6 . (canceled)  
   
   
       7 . (canceled)  
   
   
       8 . The host-vector system of  claim 4 , wherein the host-vector system comprises two vectors, and wherein the desired gene product and the essential gene on one vector is different from the desired gene product and the essential gene on the other vector.  
   
   
       9 . (canceled)  
   
   
       10 . The host-vector system of  claim 1 , further comprising a gene encoding a desired gene product.  
   
   
       11 . The host-vector system of  claim 10 , wherein the gene encodes an antigen.  
   
   
       12 . The host-vector system of  claim 11 , wherein the antigen is from a bacterial, viral, fungal, or parasitic pathogen.  
   
   
       13 . The host-vector system of  claim 12 , wherein the antigen is from  Eimeria,  HBV, or  streptococcus pneumoniae.    
   
   
       14 . A microorganism comprising the host-vector system of  claim 1 .  
   
   
       15 . A microorganism comprising the host-vector system of  claim 4 .  
   
   
       16 . (canceled)  
   
   
       17 . A vaccine comprising the microorganism of  claim 14 .  
   
   
       18 . A vaccine comprising the microorganism of  claim 15 .  
   
   
       19 . (canceled)  
   
   
       20 . A method for delivery of a nucleic acid vector to a eukaryotic host, which comprises administering to the eukaryotic host a microorganism of  claim 14 , wherein the eukaryotic host expresses the desired gene product.  
   
   
       21 . A method for delivery of a desired gene product to a eukaryotic host, which comprises administering to the eukaryotic host a microorganism of  claim 15 , wherein a prokaryote expresses the desired gene product.  
   
   
       22 . (canceled)  
   
   
       23 . The host-vector system of  claim 1 , wherein the eukaryotic promoter is CMV.  
   
   
       24 . The host-vector system of  claim 1 , wherein the prokaryotic activator-promoter sequence is araC P BAD .  
   
   
       25 . The host-vector system of  claim 1 , wherein the ori is pUC, pBR, p15A, pSC101, pBAC.  
   
   
       26 . The host-vector system of  claim 25 , wherein the ori is pUC.  
   
   
       27 . The host-vector system of  claim 1 , wherein the regulatable control sequence is P22 P R  or P trc .  
   
   
       28 . The host-vector system of  claim 27 , wherein the regulatable control sequence is P22 P R .  
   
   
       29 . The host-vector system of  claim 1 , wherein the repressor is C2, Lac I, or both.  
   
   
       30 . The host-vector system of  claim 29 , wherein the repressor is C2.  
   
   
       31 . The host-vector system of  claim 1 , wherein the essential gene is asd, murA, dapA, or alr.  
   
   
       32 . The host-vector system of  claim 1 , wherein the essential gene has a mutation that changes an ATG start codon to GTG or TTG.  
   
   
       33 . The host-vector system of  claim 1 , wherein the terminator sequence is rrFG.  
   
   
       34 . The host-vector system of  claim 1 , comprising at least three terminator sequences.  
   
   
       35 . The host-vector system of  claim 1 , comprising at least two essential genes.  
   
   
       36 . The host-vector system of  claim 1 , wherein the CpG sequence motif is GTCGTT, GACGTT, GACGTC, AACGTT, or AGCGCT.  
   
   
       37 . The host-vector system of  claim 1 , wherein the inducer is arabinose.  
   
   
       38 . (canceled)  
   
   
       39 . The method of  claim 20 , wherein the eukaryotic host is a vertebrate.  
   
   
       40 . The method of  claim 39 , wherein the vertebrate is a human, mouse, rat, or bird.  
   
   
       41 . The method of  claim 20 , wherein the microorganism colonizes a lymphoid tissue of the eukaryotic host.  
   
   
       42 . The method of  claim 41 , wherein the lymphoid tissue is in a liver, spleen, GALT, or mesenteric lymph node.  
   
   
       43 . The host-vector system of  claim 1 , further comprising a mutation in a gene to enhance immunogenicity, wherein the mutation is ΔendA2311, ΔrelA1123, ΔaraE25, ΔaraBAD]923, ΔaraBAD23, Δgmd-11, or Δgmd-fcl-26.  
   
   
       44 . A method of immunizing a poultry against coccidiosis, comprising 
 a. Administering to the poultry a microorganism comprising the host-vector system of  claim 11 , wherein the antigen is from  Eimeria;  and    b. Eliciting an immune response in the poultry.    
   
   
       45 . The method of  claim 44 , wherein the poultry is a chicken.  
   
   
       46 . The vaccine of  claim 17 , wherein the vector is a BAC vector.  
   
   
       47 . The host-vector system of  claim 4 , further comprising a gene encoding a desired gene product.  
   
   
       48 . The host-vector system of  claim 47 , wherein the gene encodes an antigen.  
   
   
       49 . The host-vector system of  claim 48 , wherein the antigen is from a bacterial, viral, fungal, or parasitic pathogen.  
   
   
       50 . The host-vector system of  claim 49 , wherein the antigen is from  Eimeria,  HBV, or  streptococcus pneumoniae.    
   
   
       51 . The host-vector system of  claim 4 , wherein the essential gene is asd, murA, dapA, or alr.  
   
   
       52 . The host-vector system of  claim 4 , wherein the essential gene has a mutation that changes an ATG start codon to GTG or TTG.  
   
   
       53 . The host-vector system of  claim 4 , wherein the terminator sequence is rrFG.  
   
   
       54 . The host-vector system of  claim 4 , comprising at least three terminator sequences.  
   
   
       55 . The host-vector system of  claim 4 , comprising at least two essential genes.  
   
   
       56 . The host-vector system of  claim 4 , wherein the inducer is arabinose.  
   
   
       57 . The method of  claim 21 , wherein the eukaryotic host is a vertebrate.  
   
   
       58 . The method of  claim 57 , wherein the vertebrate is a human, mouse, rat, or bird.  
   
   
       59 . The method of  claim 21 , wherein the microorganism colonizes a lymphoid tissue of the eukaryotic host.  
   
   
       60 . The method of  claim 59 , wherein the lymphoid tissue is in a liver, spleen, GALT, or mesenteric lymph node.  
   
   
       61 . The host-vector system of  claim 4 , further comprising a mutation in a gene to enhance immunogenicity, wherein the mutation is ΔendA2311, ΔrelA1123, ΔaraE25, ΔaraBAD1923, ΔaraBAD23, Δgmd-11, or Δgmd-fcl-26.  
   
   
       62 . A method of immunizing a poultry against coccidiosis, comprising 
 a. Administering to the poultry a microorganism comprising the host-vector system of  claim 48 , wherein the antigen is from  Eimeria;  and    b. Eliciting an immune response in the poultry.    
   
   
       63 . The method of  claim 62 , wherein the poultry is a chicken.  
   
   
       64 . The vaccine of  claim 18 , wherein the vector is a BAC vector.